The association between hepatocellular carcinoma (HCC) and chronic infection with the human hepatitis B virus (HBV) has generated considerable interest in understanding how infection with HBV increases the risk for liver cancer. Based on studies in woodchucks infected with the woodchuck hepatitis virus, it is assumed that the X protein (HBx) of all mammalian hepadnaviruses, including human HBV, is essential for viral replication. There is also evidence that HBx contributes to the development of HCC. HBx activates many cellular signaling cascades; however, how this is accomplished is unclear. Most studies that sought to identify the essential role of HBx during HBV replication, and understand how HBx regulates cellular signaling pathways, were performed in liver cell lines derived from liver tumors. The use of immortalized or transformed hepatocytes suffers from its lack of biological relevance compared to authentic hepatocytes. We have demonstrated that a key activity of HBx is regulation of cellular calcium signaling pathways. This proposal aims to understand key activities of HBx using cultures of primary rat hepatocytes and to determine how these activities influence hepatocyte physiology and HBV replication. Studies in AIM 1 will characterize the molecular mechanism of HBx activation of calcium signaling pathways in hepatocytes and how this influences HBV replication. Studies in AIM 2 will focus on the interaction between HBx and mitochondria and how this impacts hepatocyte physiology and HBV replication. AIM 3 specifically focuses on regulation of apoptosis by HBx expressed alone or during HBV replication. Although the fundamental activities of HBx are likely preserved in hepatocytes as compared to transformed cells, the magnitude, duration and complexity of these activities may differ in primary hepatocytes. Result from studies in primary rat hepatocytes should more accurately reflect the state of an authentic infected cell, clarify the disparate HBx activites that have been reported, and provide a more sophisticated understanding of the role of HBx during HBV replication.